Stereoselective synthesis of 3,7-diarylaminocholestanes by titanium-mediated reductive amination

Article abstract of DOI:10.1016/j.steroids.2014.06.018

An efficient method for the synthesis of aryl aminocholestanes, using a chlorotriisopropoxytitanium(IV)-mediated reductive amination reaction of 5α-cholestane-3,7-dione, is reported. A series of 3,7- diarylaminocholestane derivatives were prepared according to this methodology in up to 98% yield. These compounds were primarily characterized by ¹H NMR, ¹³C NMR, and mass spectrometry.

Full text of DOI:10.1016/j.steroids.2014.06.018

Steroids 88 (2014) 53–59
Contents lists available at ScienceDirect
Steroids
journal homepage: www.elsevier.com/locate/steroids
Stereoselective synthesis of 3,7-diarylaminocholestanes
by titanium-mediated reductive amination
⇑
Md. Wasi Ahmad, Hong-Seok Kim
Department of Applied Chemistry, Kyungpook National University, Daegu 702-701, Republic of Korea
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 2 May 2014
Received in revised form 11 June 2014
Accepted 20 June 2014
Available online 4 July 2014
An efficient method for the synthesis of aryl aminocholestanes, using a chlorotriisopropoxytitanium
(IV)-mediated reductive amination reaction of 5a-cholestane-3,7-dione, is reported. A series of 3,
7-diarylaminocholestane derivatives were prepared according to this methodology in up to 98% yield.
These compounds were primarily characterized by 1H NMR, 13C NMR, and mass spectrometry.
Ó 2014 Elsevier Inc. All rights reserved.
Keywords:
Reductive amination
Stereoselective reaction
Chlorotriisopropoxytitanium(IV)
3a,7a-Diarylaminocholestane
1. Introduction
is of high importance in medicinal chemistry. Moreover,
stereoselective reduction has been a major focus in synthetic
Steroids are biologically and pharmaceutically important com-
pounds and are involved in many physiological activities. They
are widely found in nature and display a variety of biological
activities [1–4]. Owing to their diverse biological properties and
wide applications, the synthesis of steroidal compounds has
gained much attention. For many years, the preparation of
aminosteroidal compounds has attracted considerable attention
from medicinal and synthetic organic chemists [5–12].
Cholesterol is an essential metabolite required for major biologi-
cal functions, such as formation the cell membrane structure
where the steroid combines with phospholipid molecules, the
integral part of the lipid bilayers [13]. However, cholesterol not
only plays a role in the physical structure of the membrane
(e.g., its viscosity and interference with the phospholipids), it also
has specific functions within the membrane. For example, the
caveolae (detergent-resistant domains) are important for trans-
duction and ceramide-induced apoptosis. It is also essential in
embryonic development, and cholesterol deficiency during
embryogenesis and organogenesis cause severe abnormalities [14].
Reductive amination of aldehydes and ketones is an impor-
tant reaction for the synthesis of amines. It is a useful tool for
the introduction of a nitrogen atom to intermediates. Chiral
amines are key features in a multitude of biologically active nat-
ural products and pharmaceuticals, and therefore their synthesis
chemistry for many decades and has been the subject of numer-
ous and diverse studies. The introduction of amino groups to the
cholesterol framework by reductive amination has been reported
by us [5–7] and others [8–10], and the products were evaluated
for biological potency and molecular recognition. These modified
steroid molecules were found to have enhanced hydrophilicity
and flexibility, which assists in intramolecular hydrogen bonding
and solubility in polar solvents, and also could be manipulated
for further transformations [15]. The preferred method to
introduce the amine group was via a Ti(OiPr)4–NaBH4 mediated
reaction, forming the 3b isomer as the major product [8–10,16].
We have also described a highly stereoselective procedure for
the synthesis of 3
oid and high-yielding, stereoselective sequential reductive
amination procedure for the synthesis of 3 ,7 -dialkylamino-
a-alkylaminosteroid [5,6], 7a-alkylaminoster-
a
a
a
cholestane [7,15]. However, these methods are only applicable
for the synthesis of aliphatic aminocholestanes. Recently,
we developed a method for one-pot reductive amination of
aromatic ketones with various aryl amines using a TiCl(OiPr)3–
NaBH(OAc)3 mediated system [17]. This protocol allows for the
synthesis of various secondary amines and 1,10-bis(N-aryl-1-
aminoethyl)ferrocene derivatives in yields up to 82%. With a reliable
method for the reductive amination of 1,1-diacetylferrocene
with aryl amines, we turned our attention to the synthesis of
aryl aminocholestanes.
The 5
a-skeleton of cholestane provides more space between the
⇑
Corresponding author. Tel.: +82 53 9505588; fax: +82 53 9506594.
E-mail address: kimhs@knu.ac.kr (H.-S. Kim).
functionalized positions at C-3 and C-7. The introduction of 3- and
http://dx.doi.org/10.1016/j.steroids.2014.06.018
0039-128X/Ó 2014 Elsevier Inc. All rights reserved.

54
M.W. Ahmad, H.-S. Kim / Steroids 88 (2014) 53–59
7-axial amine groups provide two necessary inward-pointing
hydrogen bonding sites [15,18–22], which is not possible for
cholestanes with 5b-configuration [23]. We have synthesized
various alkyl aminocholestanes with urea [15] and imidazole
[18–22] as recognizing pendants at the 3- and 7-postions of
21.8, 22.7, 22.9, 23.9, 25.1, 28.1, 28.5, 28.7, 35.2, 35.7, 36.2, 36.4,
36.9, 38.8, 39.6, 42.6, 42.7, 46.2, 47.7, 49.0, 50.2, 55.1, 55.5,
114.6, 129.5, 212.0; HR-FAB MS calcd. for C33H51NO (M+H)+:
478.4049, Found: m/z 478.4047.
5a-cholestane, as well as various alkyl aminocholestanes, for
2.2. 3
a,7a
-Di(phenylamino)-5a-cholestane (3a) and 3b,7a-
biological activity studies [24–26]. Considering the importance of
alkyl aminocholestanes, the design and synthesis of well-organized
aryl aminocholestanes for use as molecular receptors in biological
systems is highly desirable. The aryl amine pendant at C-3 and
di(phenylamino)-5
a
-cholestane (3b)
A mixture of 5a-cholestane-3,7-dione (1, 200 mg, 0.5 mmol),
1.0 M TiCl(OiPr)3 in hexane (1.2 mL, 1.2 mmol) and aniline
(140 mg, 1.5 mmol) in dry CH2Cl2 (10 mL) was stirred at room tem-
perature for 30 min. NaBH(OAc)3 (212 mg, 1.0 mmol) was then
added and the resulting mixture was stirred for an additional 1 h.
After the reaction was completed, the solvent was removed. The
residue was neutralized with NaHCO3 solution, and extracted with
ethyl acetate. The organic layer was dried and concentrated. The
residue was purified by MPLC (SiO2, elution with 0.5% EtOAc in
hexane) to give a less polar isomer 3a and more polar isomer 3b
with a 90% total yield.
C-7 of 5a-cholestane provides a more rigid frame for molecular
recognition compared to an alkyl amine pendant. Therefore, it
would be interesting to introduce an aryl amine at the C-3 and
C-7 position of the steroidal skeleton stereoselectively. Herein,
we report for the first time, an efficient method for the introduc-
tion of aryl amine into the cholesterol framework stereoselectively
through
reaction.
a
one-pot TiCl(OiPr)3-mediated reductive amination
2. Experimental section
3a: Yield 72%; amorphous; TLC Rf 0.56 (10% EtOAc in hexane);
1H NMR d 0.72 (3H, s, H-18), 0.89 (3H, d, J = 6.6 Hz, H-26), 0.90
(3H, d, J = 6.6 Hz, H-27), 0.92 (3H, s, H-19), 0.95 (3H, d, J = 6.6 Hz,
Melting points were determined using a Thomas-Hoover capil-
lary melting point apparatus and are uncorrected. NMR spectra
were recorded on a Bruker AM-400 spectrometer in CDCl3 using
Me4Si as the internal standard. High-resolution mass spectrometry
data were obtained from the Korean Basic Science Institute (Daegu
Branch) on a Jeol JMS 700 high resolution mass spectrometer. TLC
analyses were carried out on a 0.2-mm HPTLC silica gel 60 plate
and the compounds were detected by spraying with 5% ammo-
nium molybdate in 10% H2SO4 followed by heating. Flash column
chromatography was performed using Merck silica gel 60 (70–
230 mesh) and the two isomers were separated by MPLC. Reactions
were carried out under an argon atmosphere, and the solutions
were washed with brine and dried over anhydrous sodium sulfate.
H-21), 1.85 (1H, br, 7a-NH), 2.02 (1H, br t, 3a-NH), 3.54 (1H, br
s, 7b-H), 3.66 (1H, br s, 3b-H), 6.62 (4H, d, J = 7.6 Hz), 6.68 (2H,
dd, J = 7.6, 7.8 Hz), 7.15 (2H, d, J = 7.1 Hz), 7.19 (2H, d, J = 7.1 Hz);
13C NMR d 10.9, 12.0, 18.8, 20.9, 22.7, 22.9, 23.5, 23.9, 25.2, 25.4,
28.0, 28.1, 32.8, 32.9, 33.9, 35.9, 36.2, 36.5, 38.2, 39.6, 42.6, 42.7,
46.0, 47.3, 49.8, 51.6, 56.4, 112.6, 113.4, 116.5, 129.3, 129.4,
148.0; HR-FAB MS calcd. for C39H58N2 (M+H)+: 555.4678, Found:
m/z 555.4675.
3b: Yield 18%; amorphous; TLC Rf 0.43 (10% EtOAc in hexane);
1H NMR: d 0.70 (3H, s, H-18), 0.86 (3H, d, J = 6.6 Hz, H-26), 0.87
(3H, d, J = 6.6 Hz, H-27), 0.88 (3H, s, H-19), 0.92 (3H, d, J = 6.6 Hz,
H-21), 1.99 (2H, br m, 3b-NH, 7a-NH), 3.23 (1H, br m, 3a-H),
Reagents were purchased from Aldrich Chemical Co. 5
tane-3,7-dione 1 [7] and sodium tris(ethylhexanoic)borohydride
were prepared according to literature procedures [5].
a
-Choles-
3.50 (1H, br s, 7b-H), 6.57 (4H, d, J = 7.8 Hz), 6.68 (2H, dd, J = 7.6,
7.8 Hz), 7.15 (2H, d, J = 7.6 Hz), 7.17 (2H, d, J = 7.6 Hz); 13C NMR d
11.8, 12.0, 18.8, 21.3, 22.7, 22.9, 23.5, 23.9, 28.0, 28.1, 28.9, 32.7,
34.9, 35.8, 36.1, 36.2, 37.6, 38.2, 39.0, 39.6, 39.6, 42.6, 42.7, 47.4,
49.6, 51.6, 56.4, 112.5, 114.8, 116.5, 129.4, 129.4, 147.8; HR-FAB
MS calcd. for C39H58N2 (M+H)+: 555.4678, Found: m/z 555.4681.
2.1. 3
a-Phenylamino-5a-cholestan-7-one (2a) and 3b-phenylamino-
5a
-cholestan-7-one (2b)
A mixture of 5
a
-cholestane-3,7-dione (1, 200 mg, 0.5 mmol),
2.3. 3
a
,7a-Di[(4-methylphenyl)amino]-5a-cholestane (3c) and 3b,7a-
1.2 M TiCl(OiPr)3 in hexane (0.6 mL, 0.6 mmol) and aniline
(70 mg, 0.75 mmol) in methanol (10 mL) was stirred at room tem-
perature for 30 min. NaBH(OAc)3 (106 mg, 0.5 mmol) was then
added and the resulting mixture was stirred for an additional 2 h.
After the reaction was completed, the solvent was removed. The
residue was neutralized with NaHCO3 solution, and extracted with
ethyl acetate. The organic layer was dried and concentrated. The
residue was purified by MPLC (SiO2, elution with 1% EtOAc in hex-
ane) to give a less polar isomer 2a and more polar isomer 2b with
an 80% total yield.
2a: Yield 64%; amorphous; TLC Rf 0.35 (10% EtOAc in hexane);
1H NMR d 0.66 (3H, s, H-18), 0.86 (3H, d, J = 6.6 Hz, H-26), 0.87
(3H, d, J = 6.6 Hz, H-27), 0.91 (3H, d, J = 6.6 Hz, H-21), 1.09 (3H, s,
H-19), 3.71 (1H, br s, 3b-H), 6.69 (2H, d, J = 7.8 Hz), 6.76 (1H, t,
J = 7.3 Hz), 7.18 (2H, dd, J = 7.3, 7.8 Hz); 13C NMR d 11.1, 12.2,
18.9, 21.3, 22.7, 22.9, 23.9, 25.1, 25.6, 28.1, 28.5, 32.3, 32.7, 35.7,
36.2, 36.6, 38.8, 39.6, 42.6, 42.7, 46.0, 47.7, 49.0, 50.3, 55.1, 55.8,
114.8, 129.4, 212.1; HR-FAB MS calcd. for C33H51NO (M+H)+:
478.4049, Found: m/z 478.4053.
di[(4-methylphenyl)-amino]-5
a
-cholestane (3d)
3c: Yield 82%; amorphous; TLC Rf 0.54 (10% EtOAc in hexane);
1H NMR d 0.72 (3H, s, H-18), 0.89 (3H, d, J = 6.8 Hz, H-26), 0.90
(3H, d, J = 6.6 Hz, H-27), 0.90 (3H, s, H-19), 0.95 (3H, d, J = 6.6 Hz,
H-21), 2.00 (1H, m, 7a-NH), 2.03 (1H, m, 3a-NH), 2.25 (3H, s,
CH3), 2.26 (3H, s, CH3), 3.51 (1H, br s, 7b-H), 3.62 (1H, br s, 3b-
H), 6.56 (4H, d, J = 6.6 Hz), 6.97 (2H, d, J = 7.8 Hz), 6.99 (2H, d,
J = 7.8 Hz); 13C NMR d 11.2, 12.4, 19.1, 20.7, 20.8, 20.8, 21.2, 23.0,
23.2, 23.8, 24.2, 25.6, 28.1, 28.4, 32.7, 33.0, 34.0, 36.2, 36.5, 36.7,
38.5, 39.8, 39.9, 43.70, 47.5, 50.3, 51.9, 54.1, 56.7, 113.1, 125.8,
130.1, 130.2, 146.1; HR-FAB MS calcd. for
582.4913, Found: m/z 582.4916.
3d: Yield 14%; amorphous, TLC Rf 0.40 (10% EtOAc in hexane);
1H NMR d 0.67 (3H, s, H-18), 0.85 (3H, d, J = 6.6 Hz, H-26), 0.86
(3H, d, J = 6.6 Hz, H-27), 0.86 (3H, s, H-19), 0.90 (3H, d, J = 6.3 Hz,
C
41H62N2 (M+):
H-21), 1.96, (1H, m, 3b-NH), 1.99 (1H, m, 7
CH3), 2.24 (3H, s, CH3), 3.15 (1H, br m, 3 -H), 3.44 (1H, br s,
a-NH), 2.22 (3H, s,
a
7b-H), 6.48 (2H, d, J = 8.4 Hz), 6.75 (2H, d, J = 6.8 Hz), 6.96 (2H, d,
J = 8.1 Hz), 6.99 (2H, d, J = 8.1 Hz); 13C NMR d 12.1, 12.4, 19.1,
20.7, 21.0, 21.6, 23.0, 23.2, 23.8, 24.2, 25.6, 28.4, 28.5, 32.9, 34.0,
36.2, 36.5, 36.6, 37.8, 38.5, 39.1, 39.9, 40.0, 43.0, 47.6, 50.0, 51.9,
54.1, 56.7, 112.9, 125.9, 130.2, 130.3, 145.8; HR-FAB MS calcd. for
2b: Yield 16%; amorphous; TLC Rf 0.19 (10% EtOAc in hexane);
1H NMR d 0.66 (3H, s, H-18), 0.86 (3H, d, J = 6.6 Hz, H-26), 0.87
(3H, d, J = 6.6 Hz, H-27), 0.92 (3H, d, J = 6.6 Hz, H-21), 1.09 (3H, s,
H-19), 3.26 (1H, m, 3
a-H), 6.61 (2H, d, J = 7.6 Hz), 6.70 (1H, t,
J = 7.1 Hz), 7.16 (2H, t, J = 7.6 Hz); 13C NMR d 12.0, 12.2, 18.9,
C
41H62N2 (M+): 582.4913, Found: m/z 582.4918.

M.W. Ahmad, H.-S. Kim / Steroids 88 (2014) 53–59
55
2.4. 3
3b,7
a
,7
a
-Di[(4-methoxyphenyl)amino]-5
a
-cholestane (3e) and
calcd. for C39H56N2Br2 (M+H)+, (M+2)+ and (M+4)+: 711.2888,
712.2922 and 714.2902, Found: m/z 711.2888, 712.2947 and
714.2902.
a-[di(4-methoxy-phenyl)amino]-5
a-cholestane (3f)
3e: Yield 88%; amorphous; TLC Rf 0.5 (25% EtOAc in hexane); 1H
NMR d 0.69 (3H, s, H-18), 0.87 (3H, d, J = 6.6 Hz, H-26), 0.87 (3H, d,
J = 6.6 Hz, H-27), 0.87 (3H, s, H-19), 0.92 (3H, d, J = 6.6 Hz, H-21),
3j: Yield 22%; amorphous, TLC Rf 0.35 (10% EtOAc in hexane); 1H
NMR d 0.68 (3H, s, H-18), 0.85 (3H, d, J = 6.6 Hz, H-26), 0.86 (3H, d,
J = 6.6 Hz, H-27), 0.86 (3H, s, H-19), 0.91 (3H, d, J = 6.3 Hz, H-21),
1.99 (1H, d, J = 12.6 Hz, 7
a-NH), 3.44 (1H, br s, 7b-H), 3.55 (1H,
1.96, (1H, br d, 3b-NH), 2.00 (1H, br s, 7
a-NH), 3.17 (1H, m,
br s, 3b-H), 3.73 (3H, s, OCH3), 3.74 (3H, s, OCH3), 6.58 (2H, d,
J = 7.6 Hz), 6.64 (2H, br s), 6.72 (2H, d, J = 8.8 Hz), 6.76 (2H, d,
J = 9.1 Hz); 13C NMR d 11.0, 12.0, 18.8, 20.9, 22.7, 22.9, 23.6, 23.9,
25.4, 28.1, 29.4, 29.9, 32.6, 32.7, 33.3, 35.9, 36.2, 36.4, 38.2, 38.3,
39.6, 39.6, 42.7, 47.2, 50.9, 51.5, 55.8, 56.0, 56.3, 114.2, 115.0,
115.1, 138.7, 142.4, 151.5; HR-FAB MS calcd. for C41H62N2O2
(M+H)+: 615.4890, Found: m/z 615.4886.
3a-H), 3.42 (1H, br s, 7b-H), 6.44 (2H, d, J = 8.8 Hz), 6.53 (2H, d,
J = 8.3 Hz), 7.22 (2H, d, J = 8.6 Hz), 7.24 (2H, d, J = 8.6 Hz); 13C
NMR d 11.8, 12.0, 18.4, 18.8, 21.3, 22.7, 23.0, 23.5, 24.0, 25.1,
28.0, 28.1, 28.7, 32.5, 34.8, 35.8, 36.1, 36.2, 37.5, 38.1, 39.0, 39.5,
42.7, 47.4, 49.8, 51.6, 56.3, 107.9, 114.1, 114.2, 116.3, 132.1,
132.2, 141.8, 146.7; HR-FAB MS calcd. for C39H56N2Br2 (M+H)+,
(M+2)+ and (M+4)+: 711.2888, 712.2922 and 714.2902, Found: m/
z 711.2892, 712.2941 and 714.2950.
3f: Yield 10% amorphous; TLC Rf 0.5 (25% EtOAc in hexane); 1
H
NMR d 0.69 (3H, s, H-18), 0.86 (3H, d, J = 6.3 Hz, H-26), 0.87 (3H, d,
J = 6.5 Hz, H-27), 0.87 (3H, s, H-19), 0.92 (3H, d, J = 6.3 Hz, H-21),
2.8. 3
a,7
a-Di[(3-bromophenyl)amino]-5a-cholestane (3k) and 3b,
3.15 (1H, br m, 3
a-H), 3.35 (1H, br s, 7
a-NH), 3.41 (1H, br s,
7a
-di[(3-bromophenyl)-amino]-5a
-cholestane (3l)
7b-H), 3.62 (1H, br t, 3b-NH), 3.73 (3H, s, OCH3), 3.75 (3H, s,
OCH3), 6.54 (2H, d, J = 9.0 Hz), 6.58 (2H, s), 6.75 (2H, d,
J = 8.8 Hz), 6.77 (2H, d, J = 8.8 Hz); 13C NMR d 11.8, 12.0, 18.8,
21.3, 22.6, 22.9, 23.5, 23.8, 28.0, 28.1, 29.5, 29.8, 32.8, 35.6, 35.8,
36.2, 37.7, 38.3, 38.9, 39.5, 39.6, 42.7, 47.3, 50.6, 51.5, 54.0, 55.8,
55.9, 56.3, 113.8, 115.0, 115.1, 115.5, 128.3, 130.1, 141.1, 142.3,
3k: Yield 63%; amorphous; TLC Rf 0.59 (10% EtOAc in hexane);
1H NMR d 0.69 (3H, s, H-18), 0.87 (3H, d, J = 6.6 Hz, H-26), 0.87
(3H, d, J = 6.8 Hz, H-27), 0.88 (3H, s, H-19), 0.92 (3H, d, J = 6.3 Hz,
H-21), 2.00 (1H, br s, 7
a-NH), 3.47 (1H, br s, 7b-H), 3.60 (1H, br
s, 3b-H), 3.97 (1H, br s, 3
a-NH), 6.48 (2H d, J = 6.8 Hz), 6.74 (4H,
151.5, 152.3; HR-FAB MS calcd. for
C
41H62N2O2 (M+H)+:
m), 6.99 (2H, dd, J = 8.1, 8.1 Hz); 13C NMR d 10.9, 12.0, 18.7, 22.7,
22.9, 23.4, 23.8, 25.3, 28.0, 28.1, 32.5, 32.8, 34.0, 35.4, 35.5, 36.1,
36.4, 37.9, 39.5, 39.6, 42.7, 47.3, 47.5, 49.7, 54.2, 51.6, 56.3,
111.1, 111.6, 114.4, 114.9, 119.2, 119.4, 123.4, 123.5, 130.5,
130.7, 148.7, 149.1; HR-FAB MS calcd. for C39H56N2Br2 (M+H)+,
(M+2)+ and (M+4)+: 711.2888, 712.2922 and 714.2902, Found: m/
z 711.2892, 712.2922 and 714.2950.
615.4890, Found: m/z 615.4886.
2.5. 3a,7a-Di[(2-aminophenyl)amino]-5a-cholestane (3g)
3g: Yield 80% amorphous; TLC Rf 0.5 (25% EtOAc in hexane); 1H
NMR d 0.70 (3H, s, H-18), 0.86 (3H, d, J = 6.6 Hz, H-26), 0.87 (3H, d,
J = 6.8 Hz, H-27), 0.90 (3H, s, H-19), 0.92 (3H, d, J = 6.5 Hz, H-21),
3l: Yield 21%; amorphous; TLC Rf 0.32 (10% EtOAc in hexane);
1H NMR d 0.68 (3H, s, H-18), 0.86 (3H, d, J = 6.6 Hz, H-26), 0.87
(3H, d, J = 6.8 Hz, H-27), 0.87 (3H, s, H-19), 0.91 (3H, d, J = 6.3 Hz,
3.23 (1H, m, 7
3.93 (4H, br s, NH2), 2.03 (1H, m, 3
a
-NH), 3.58 (1H, br s, 7b-H), 3.69 (1H, br s, 3b-H),
-NH), 6.56 (4H, d, J = 6.6 Hz),
a
6.97 (2H, d, J = 7.8 Hz), 6.99 (2H, d, J = 7.8 Hz); 13C NMR d 11.0,
11.8, 12.0, 12.1, 18.7, 20.9, 21.3, 22.6, 22.9, 23.5, 23.6, 23.9, 28.0,
28.1, 28.7, 32.1, 32.2, 35.8, 35.9, 36.2, 36.3, 36.4, 37.5, 38.3, 38.4,
38.9, 39.6, 42.8, 47.3, 49.0, 51.6, 51.7, 56.2, 56.3, 110.5, 117.1,
117.2, 117.3, 120.5, 121.3, 133.7, 137.7; HR-FAB MS calcd. for
H-21), 2.00 (1H, br s, 7
a-NH), 2.03 (1H, br d, 3b-NH), 3.19 (1H, br
m, 3 -H), 3.45 (1H, br s, 7b-H), 4.01 (1H, br s, 3
a
a
-NH), 6.49 (2H,
t, J = 8.6 Hz), 6.73 (4H, m), 6.99 (2H, dd, J = 7.8, 7.8 Hz); 13C NMR
d 11.8, 12.0, 18.7, 21.2, 22.7, 22.9, 23.4, 23.8, 28.0, 28.1, 28.9,
32.4, 35.1, 35.8, 36.0, 36.1, 37.5, 38.0, 39.0, 39.5, 39.6, 42.7, 47.4,
49.5, 51.5, 52.9, 56.3, 111.1, 112.4, 115.0, 116.3, 119.2, 120.4,
123.3, 123.5, 130.6, 130.7, 147.9, 149.0; HR-FAB MS calcd. for
C
39H60N4 (M+): 584.4820, Found: m/z 584.4822.
2.6. 3 ,7 -Di[(4-pyridyl)amino]-5 -cholestane (3h)
a
a
a
C
39H56N2Br2 (M+H)+, (M+2)+ and (M+4)+: 711.2888, 712.2922 and
714.2902, Found: m/z 711.2893, 712.2951 and 714.2942.
3h: Yield 80% amorphous; TLC Rf 0.51 (30% EtOAc in hexane);
1H NMR d 0.78 (3H, s, H-18), 0.82 (3H, d, J = 6.6 Hz, H-26), 0.85
(3H, d, J = 6.8 Hz, H-27), 0.88 (3H, s, H-19), 0.90 (3H, d, J = 6.5 Hz,
2.9. 3
a
,7
a
-Di[(2-bromophenyl)amino]-5
a-cholestane (3m) and
3a
-(2-bromophenyl)amino-5a
-cholestan-7-one (2c)
H-21), 2.01 (1H, br s, 7a-NH), 2.03 (1H, br s, 3a-NH), 3.56 (1H, br
s, 7b-H), 3.69 (1H, br s, 3b-H), 6.45 (4H, d, J = 7.0 Hz), 8.02 (4H, d,
J = 7.0 Hz); 13C NMR d 11.1, 12.0, 18.6, 20.7, 21.4, 22.9, 23.0, 23.2,
23.6, 24.6, 28.0, 28.2, 28.7, 32.1, 32.3, 35.2, 35.7, 36.2, 36.4, 37.9,
38.4, 38.5, 38.9, 39.6, 42.9, 47.0, 51.6, 51.7, 56.2, 56.3, 112.5,
112.8, 148.6, 148.7, 155.6; HR-FAB MS calcd. for C37H56N4 (M+):
556.4521, Found: m/z 556.4523.
A mixture of 5a-cholestane-3,7-dione (1, 200 mg, 0.5 mmol),
1.0 M TiCl(OiPr)3 in hexane (1.2 mL, 1.2 mmol) and 2-bromoaniline
(140 mg, 1.5 mmol) in dry CH2Cl2 (10 mL) was stirred at room tem-
perature for 30 min. NaBH(OEh)3 (212 mg, 1.0 mmol) was then
added and the resulting mixture was stirred for an additional
30 h. After the reaction was completed, the solvent was removed.
The residue was neutralized with NaHCO3 solution, and extracted
with ethyl acetate. The organic layer was dried and concentrated.
The residue was purified by MPLC (SiO2, elution with 0.5% EtOAc
in hexane) to give a less polar compound 3l and more polar com-
pound 2c.
2.7. 3
3b,7
a
,7
a
-Di[(4-bromophenyl)amino]-5
a-cholestane (3i) and
a-di[(4-bromo-phenyl)amino]-5
a-cholestane (3j)
3i: Yield 68%; amorphous; TLC Rf 0.51 (10% EtOAc in hexane);
1H NMR d 0.69 (3H, s, H-18), 0.87 (3H, d, J = 6.8 Hz, H-26), 0.88
(3H, d, J = 6.8 Hz, H-27), 0.88 (3H, s, H-19), 0.92 (3H, d, J = 6.6 Hz,
3m: Yield 65%; amorphous; TLC Rf 0.67 (10% EtOAc in hexane);
1H NMR d 0.71 (3H, s, H-18), 0.88 (3H, d, J = 6.5 Hz, H-26), 0.89 (3H,
d, J = 6.5 Hz, H-27), 0.93 (3H, s, H-19), 0.94 (3H, d, J = 6.3 Hz, H-21),
H-21), 1.99 (1H, br s, 7a-NH), 2.02 (1H, br s, 3a-NH), 3.44 (1H, br
s, 7b-H), 3.58 (1H, br s, 3b-H), 6.46 (4H, d, J = 8.3 Hz), 7.21 (2H, d,
J = 8.8 Hz), 7.22 (2H, d, J = 8.8 Hz); 13C NMR d 10.9, 12.0, 18.5,
18.8, 20.9, 22.7, 23.0, 23.5, 24.0, 25.3, 28.0, 28.1, 32.6, 32.8, 33.9,
35.8, 36.2, 36.4, 38.1, 38.6, 39.6, 42.8, 47.3, 50.0, 51.6, 56.4,
107.8, 114.1, 114.4, 115.0, 125.5, 132.0, 132.1, 147.0; HR-FAB MS
2.01 (1H, m, 7
a-NH), 3.62 (1H, br s, 7b-H), 3.7 (1H, br s, 3b-H), 4.73
(1H, br s, 3 -NH), 6.49 (1H, dt, J = 7.6, 1.8 Hz), 6.51 (1H, dt, J = 7.6,
a
1.8 Hz), 6.58 (2H, d, J = 8.3 Hz), 7.12 (1H, dt, J = 7.6, 1.8 Hz), 7.14
(1H, dt, J = 7.6, 1.8 Hz), 7.40 (1H, d, J = 8.3 Hz), 7.41 (1H, d,
J = 8.3 Hz); 13C NMR d 11.0, 12.1, 18.8, 20.9, 22.7, 22.9, 23.4, 23.5,

56
M.W. Ahmad, H.-S. Kim / Steroids 88 (2014) 53–59
23.9, 25.1, 28.0, 28.1, 32.5, 32.6, 34.2, 35.9, 36.2, 36.5, 38.2, 39.5,
39.6, 42.7, 47.3, 47.9, 49.9, 51.6, 56.2, 110.5, 110.7, 111.0, 111.8,
116.9, 117.2, 128.4, 128.5, 132.6, 132.6, 143.9, 144.4; HR-FAB MS
calcd. for C39H56N2Br2 (M+H)+, (M+2)+ and (M+4)+: 711.2888,
712.2922 and 714.2902, Found: m/z 711.2881, 712.2930 and
714.2927.
42.8, 47.5, 49.8, 51.6, 56.3, 107.4, 111.0, 111.3, 126.6, 126.8,
137.7, 152.4, 152.7; HR-FAB MS calcd. for C39H56N4O4 (M)+:
645.4380, Found: m/z 645.4377.
3. Results and discussion
2c: Yield 11%; white solid; mp 82–84 °C (CH2Cl2–CH3CN); TLC Rf
0.32 (10% EtOAc in hexane); 1H NMR d 0.66 (3H, s, H-18), 0.85 (3H,
d, J = 6.5 Hz, H-26), 0.87 (3H, d, J = 6.5 Hz, H-27), 0.91 (3H, d,
J = 6.5 Hz, H-21), 1.10 (3H, s, H-19), 3.77 (1H, br s, 3b-H), 4.77
Recently, we reported a sequential stepwise procedure for the
synthesis of highly stereoselective 3
tanes, starting from 5 -cholestane-3,7-dione (1), in good yields of
up to 84% [15]. Unfortunately, this methodology was not applicable
a,7a-dialkylamino-5a-choles-
a
(1H, br s, 3a-NH), 6.51–6.57 (1H, m), 6.63 (1H, d, J = 8.0 Hz), 7.14
(1H, dt, J = 8.0, 1.2 Hz), 7.40 (1H, d, J = 8.0 Hz); 13C NMR d 11.1,
12.2, 18.9, 21.3, 22.7, 22.9, 23.9, 25.1, 25.6, 28.1, 28.5, 32.3, 32.8,
35.8, 36.2, 36.6, 38.8, 39.6, 42.6, 42.8, 46.0, 47.8, 49.0, 50.3, 55.1,
55.8, 110.6, 112.3, 118.0, 128.5, 132.7, 143.2, 211.8; HR-FAB MS
calcd. for C33H50BrNO (M+H)+ and (M+2)+: 557.2808, 558.2808
Found: m/z 557.2871 and 558.2871.
to the synthesis of 3,7-diarylamino-5a-cholestanes. Using an effi-
cient TiCl(OiPr)3-mediated reductive amination [17,27,28], we
have envisioned a one-step procedure for the preparation of new
3,7-diarylamino-5a-cholestane derivatives from 5a-cholestane-
3,7-dione (1) according to Scheme 1. This study initially examined
the reductive amination reaction using 1 and aniline, with the
results presented in Table 1.
2.10. 3
a
,7a
-Di[(4-carbomethoxyphenyl)amino]-5a-cholestane (3n)
It was found that the yields of 3,7-diphenylamino-5a-choles-
and 3b,7
a
-di[(4-4-carbomethoxyphenyl)amino]-5
a
-cholestane (3o)
tane (3) depended on the solvent and titanium source used. Dichlo-
romethane faired best among the test solvents and desired product
3 was isolated in 90% yield using a TiCl(OiPr)3-mediated reaction at
room temperature (Table 1, entry 4). When the same reaction was
3n: Yield 63%; amorphous; TLC Rf 0.59 (10% EtOAc in hexane);
1H NMR d 0.66 (3H, s, H-18), 0.84 (3H, d, J = 6.6 Hz, H-26), 0.85
(3H, d, J = 6.6 Hz, H-27), 0.87 (3H, s, H-19), 0.89 (3H, d, J = 6.5 Hz,
carried out in MeOH, 3-phenlyamino-5a-cholestan-7-one (2) was
H-21), 1.97 (1H, d, J = 12.6, 7
(1H, br s, 3b-H), 3.82 (3H, s, CH3), 3.83 (3H, s, CH3), 4.52 (1H, br
a
-NH), 3.55 (1H, br s, 7b-H), 3.68
isolated with 80% yield (Table 1, entry 5). To evaluate the influence
of the titanium source as a Lewis acid, the reaction was performed
using acetic acid in CH2Cl2. A mixture of 2 (29–36%) and 3 (47–
53%) was isolated from the direct and indirect reductive amination,
respectively (Table 1, entries 1–2). Furthermore, TiCl(OiPr)3 and
Ti(OiPr)4 were investigated as the titanium source in CH2Cl2 at
room temperature (Table 1, entries 4 and 6), with the best results
obtained using TiCl(OiPr)3. Interestingly, the Ti(OiPr)4-mediated
reaction of 1 with aniline, using NaBH4 in MeOH at ꢀ78 °C, affor-
s, 3a-NH) 6.53 (4H, d, J = 8.3 Hz), 7.80 (2H, d, J = 8.8 Hz), 7.83
(2H, d, J = 8.8 Hz); 13C NMR d 10.8, 11.9, 18.7, 20.8, 22.6, 22.9,
23.3, 23.8, 25.2, 27.9, 28.0, 32.7, 34.1, 35.8, 36.1, 36.3, 37.9, 39.4,
39.5, 42.7, 47.2, 49.5, 51.5, 51.6, 56.2, 111.3, 111.5, 117.5, 131.6,
131.8, 151.0, 151.6, 167.4; HR-FAB MS calcd. for C43H62N2O4
(M+H)+: 671.4788, Found: m/z 671.4790.
3o: Yield 21%; amorphous; TLC Rf 0.32 (10% EtOAc in hexane);
1H NMR d 0.66 (3H, s, H-18), 0.83 (3H, d, J = 6.8 Hz, H-26), 0.84
(3H, d, J = 6.6 Hz, H-27), 0.85 (3H, s, H-19), 0.89 (3H, d, J = 6.3 Hz,
ded 3a-amino (2a) and 3b-amino (2b) in a 1:9 ratio and 70% over-
all yield (Table 1, entry 8) [8–10,16]. In an attempt to minimize the
formation of undesired product 2, the TiCl(OiPr)3-mediated reduc-
tive amination of 1 was carried out using aniline in CH2Cl2 and
NaBH(OAc)3 as the reducing agent at room temperature to afford
H-21), 2.16 (1H, s, 7
a-NH), 3.24 (1H, m, 3
a
-H), 3.54 (1H, br s, 7b-
-NH)
H), 3.83 (3H, s, CH3), 3.84 (3H, s, CH3), 4.45 (1H, br s, 3
a
6.63 (4H, d, J = 8.6 Hz), 7.84 (4H, d, J = 8.5 Hz); 13C NMR d 11.7,
12.0, 18.7, 21.2, 22.6, 22.9, 23.3, 23.8, 27.9, 28.1, 28.2, 29.8, 31.0,
32.5, 34.2, 35.7, 35.9, 36.1, 37.2, 37.8, 38.9, 39.4, 39.5, 42.7, 47.3,
49.3, 51.5, 51.6, 51.7, 51.8, 52.0, 56.2, 111.3, 113.9, 117.5, 117.9,
119.6, 130.9, 131.4, 131.6, 131.8, 150.9, 151.4, 167.3, 167.4; HR-
FAB MS calcd. for C43H62N2O4 (M+H)+: 671.4788, Found: m/z
671.4790.
3
a a (3a) and 3b,7a (3b) in a 4:1 ratio (Table 1, entry 4).
,7
It was hypothesized that replacing one of the isopropoxide
ligands from Ti(OiPr)4 with a chlorine atom would increase the
Lewis acidity of the reagent and facilitate the condensation of the
amine with the ketone [17,27,28]. A possible mechanism for the
nucleophilic attack of the amino group on the Lewis acid activated
carbonyl compound is shown in Scheme 2 [29,30]. In this case, the
reaction may proceed through titanium complex I, which could be
reduced directly or through transient imine species II.
2.11. 3
a
,7a
-Di[(4-nitrophenyl)amino]-5
a-cholestane (3p) and 3b,7a-
di[(4-nitrophenyl)-amino]-5a-cholestane (3q)
The stereoselective introduction of an amino group at C-3, with
3p: Yield 64%; amorphous; TLC Rf 0.80 (33% EtOAc in hexane);
1H NMR d 0.66 (3H, s, H-18), 0.83 (3H, d, J = 6.6 Hz, H-26), 0.84
(3H, d, J = 6.6 Hz, H-27), 0.88 (3H, d, J = 6.6 Hz, H-21), 0.90 (3H, s,
configuration 3a and 3b, depended on the reducing agent
selection. Smaller reagents like NaBH4 and NaBH3CN yielded the
3b isomer as the major product, as it attacks from the axial side
at C-3. Bulky reagents such as sodium triacetoxyborohydride
[NaBH(OAc)3] and sodium tris[2-(ethylhexanoic)]borohydride
[NaBH(OEh)3] [7,15,31] attack from the equatorial side at C-3
H-19), 1.97 (1H, br s, 7
s, 3b-H), 5.20 (1H, br s, 3
a
-NH), 3.62 (1H, br s, 7b-H), 3.77 (1H, br
-NH), 6.46 (4H, d, J = 8.8 Hz), 7.91 (2H,
a
d, J = 9.1 Hz), 7.98 (2H, d, J = 9.1 Hz); 13C NMR d 10.8, 11.9, 18.7,
20.8, 22.6, 22.9, 23.4, 23.9, 25.0, 27.9, 28.1, 29.8, 32.3, 32.6, 34.2,
35.8, 36.1, 36.3, 37.8, 39.4, 39.5, 42.8, 47.0, 47.8, 49.9, 51.5, 56.3,
110.8, 111.3, 126.6, 126.8, 136.9, 137.1, 152.8, 153.3; HR-FAB MS
calcd. for C39H56N4O4 (M)+: 645.4380, Found: m/z 645.4383.
3q: Yield 21%; amorphous; TLC Rf 0.68 (33% EtOAc in hexane);
1H NMR d 0.68 (3H, s, H-18), 0.84 (3H, d, J = 6.6 Hz, H-26), 0.85
(3H, d, J = 6.8 Hz, H-27), 0.89 (3H, s, H-19), 0.90 (3H, d, J = 6.6 Hz,
leading to the formation of the 3a isomer predominantly [32].
The stereochemistry of 3 was determined based on the Rf value
and the chemical shifts of the protons and carbons at the 3- and 7-
positions in the 1H and 13C NMR, respectively. The 1H NMR spec-
trum of the 3a-amino isomer (3c), peaks representing the 3b-H
and 7b-H protons appeared at 3.62 ppm (br s) and 3.51 ppm (br
s), respectively. In the 13C NMR spectrum of 3c, C-3 and C-7 car-
bons appeared at 47.5 ppm and 50.3 ppm, respectively. In compar-
H-21), 2.01 (1H, br s, 7
7b-H), 4.83 (1H, br s, 3
a-NH), 3.34 (1H, m, 3a-H), 3.63 (1H, br s,
a-NH), 6.48 (2H, d, J = 8.1 Hz), 6.53 (2H, d,
ison, peaks for the 3a-H and 7b-H of the 3b-isomer (3d) appeared
J = 9.1 Hz), 8.02 (2H, d, J = 9.1 Hz), 8.07 (2H, d, J = 8.8 Hz); 13C
NMR d 11.8, 12.0, 18.7, 21.3, 22.6, 22.9, 23.4, 23.9, 27.9, 28.1,
28.8, 29.8, 32.6, 34.9, 35.8, 36.0, 36.1, 37.3, 37.8, 39.2, 39.4, 39.5,
at d 3.15 (br m) and 3.44 (br s), respectively. For the 13C NMR spec-
trum of 3d, the C-3 and C-7 carbon appeared at d 47.6 and 50.0,
respectively. The 2D-HETCO spectrum of 3c revealed that 3b-1H

M.W. Ahmad, H.-S. Kim / Steroids 88 (2014) 53–59
57
SC
SC
SC
C6H5NH2
Activator
Step 1
+
HN
O
HN
NH
NaBH(OAc)3
Step 2
H
H
O
O
H
α
2a : 3
3a : 3α, 7α
1
2b : 3β
β,
α
7
3b : 3
SC=
Scheme 1. Initial optimization for the synthesis of 3,7-diarylamino-5a-cholestane derivatives.
Table 1
Reductive amination of 1 with aniline using NaBH(OAc)3 at room temperature.
information). The chemical shifts observed were in accordance
with those found previously [10,15,32].
Entry Activatorb Solvent Time (h) Time (h) Yieldc (%) Yieldc (%)
Step 1
Step 2
2a:2b
3a:3b
The general applicability of this procedure for the synthesis of a
diverse range of aromatic amines was then studied and the results
are presented in Table 2. The generality of this method was
evaluated using 2-, 3- and 4-substituted aniline derivatives.
Based on these results, it was observed that the presence of
substituents on aniline improved the stereoselectivity without
affecting the reactivity. The reaction of 1 with 4-methylaniline
using NaBH(OAc)3 gave the corresponding 3,7-di[(4-methyl-
1
2
3
4
5
6
7
8a
A
A
B
B
B
C
C
C
CH2Cl2
CH2Cl2
THF
CH2Cl2
MeOH
CH2Cl2
MeOH
MeOH
0
24
24
24
1
2
5
36 (2:1)
29 (2:1)
–
47 (2:1)
53 (2:1)
83 (4:1)
90 (4:1)
–
–
–
–
14
0.5
0.5
0.5
12
12
12
–
80 (4:1)
77 (4:1)
78 (4:1)
70 (1:9)
2
2
a
b
c
phenyl)amino]-5a-cholestanes as a mixture of 3c:3d (6:1) in 96%
NaBH4 at ꢀ78 °C.
A: AcOH (5 drops), B: TiCl(OiPr)3, C: Ti(OiPr)4.
Isolated yields.
yield (Table 2, entry 3). High diastereoselectivity was achieved by
modifying NaBH4 with 2-ethylhexanoic acid (Eh). NaBH(OEh)3
afforded products 3c:3d in a 36:1 ratio and 97% yield (Table 2,
entry 4). The highest stereoselectivity and yield were achieved
using 4-anisidine. Even electron-deficient anilines containing
bromo, ester, and nitro groups (Table 2, entries 10–17) reacted
with 1 to yield the corresponding secondary aryl amines. The
and 7b-1H were connected to C-3 and C-7, respectively. Further
relative stereochemistry assignment was established by data
obtained from the COSY and DEPT spectra (Supplementary
SC
SC
SC
1) C6H5NH2, Solvent
TiCl(OiPr)3, 0.5 h
HN
O
HN
NH
H
H
3
2) NaBH(OAc)3, 1h
O
O
H
1
2
TiCl(OiPr)3
SC
NaBH(OAc)3
SC
H2N
Ti(OiPr)3
Cl
HO
N
O
O
H
H-N
H
II
SC
-Ti(OiPr)3OH
(iPrO)3Ti
O
O
H-N
H
Cl
Complex I
Scheme 2. Proposed mechanism.

58
M.W. Ahmad, H.-S. Kim / Steroids 88 (2014) 53–59
Table 2
Stereoselective synthesis of 3,7-diarylaminocholestanes derivatives
SC
SC
TiCl(OiPr)3
ArNH2, CH2Cl2
Reducing agent
r.t.
O
O
ArHN
NHAr
H
1
H
3
.
Entry
Amine
Ar
Reagenta
Time
(h)
Yieldb (%)
Ratio
,7
Product
3
a
a
:3b,7
a
1
2
3
4
5
6
7
8
C6H5
C6H5
D
E
D
E
D
E
D
E
E
D
E
D
E
E
1
1
1
1
1
1
24
20
24
3
90
95
96
97
98
98
80
83
80
90
92
84
89
65c
83
85
85
4:1
10:1
6:1
36:1
8:1
100:0
100:0
100:0
100:0
3:1
14:1
3:1
9:1
3a–3b
3a–3b
3c–3d
3c–3d
3e–3f
3e
3g
3g
3h
3i–3j
3i–3j
3k–3l
3k–3l
3m
3n–3o
3n–3o
3p–3q
4-CH3C6H4
4-CH3C6H4
4-CH3OC6H4
4-CH3OC6H4
2-NH2C6H4
2-NH2C6H4
4-NC5H4
4-BrC6H4
4-BrC6H4
3-BrC6H4
3-BrC6H4
9
10
11
12
13
14
15
16
17
3
24
24
30
24
20
72
2-BrC6H4
100:0
3:1
6:1
4-CH3O2CC6H4
4-CH3O2CC6H4
4-O2NC6H4
D
E
D
3:1
a
b
c
D: NaBH(OAc)3, E: NaBH(OEh)3.
Isolated yields.
3a-(2-Bromophenyl)amino-5a-cholestan-7-one (2c) was isolated in 11%.
reaction of 1 with 2-, 3-, 4-bromoaniline proceeded slowly and
afforded the desired product in good to high yields. A marked ster-
eoselective effect was observed with respect to the position of the
bromo group of the substrate (Table 2, entries 11, 13 and 14).
Unhindered 4-bromoaniline afforded the corresponding product
in 92% yield (Table 2, entry 11). Reaction of more hindered 2-bro-
moaniline occurred at room temperature, affording the desired
product in moderate yield (65%). Steric hindrance appeared to play
a role in the outcome of the reaction as exemplified by the conver-
sion of electron-rich 2-aminoaniline (Table 2, entry 7) which pro-
duced only a single isomer in good yield even with NaBH(OAc)3.
In comparison, the reaction of 1 with 2-bromoaniline (Table 2,
S. aureus 77, S. aureus 241, Staphylococcus epidermidis 887E, Bacillus
subtilis ATCC6633 and Bacillus licheniformis EMR. None of Gram-
positive and Gram-negative bacteria were resistant to compounds
3a, 3b and 3i having MIC value >32 lg/mL.
4. Conclusion
An efficient method for the one-pot synthesis of a new class of
steroids has been developed. 3 ,7 -Diarylamino-5 -cholestane
a
a
a
derivatives were synthesized in high yields. This novel method
could be useful for a wide range of synthetic applications as it is
mild, efficient, and environmentally friendly. 3a,7a-Dipyridylami-
no compound (3h) showed moderate antimicrobial activity toward
the Gram-positive bacterias, S. aureus ATCC6538P and Micrococcus
luteus ATCC9341.
entry 14) produced only 3
a,7
a-diaryl compound (3m) in a moder-
ate yield of 65%, with 11% of the 3
a-mono aminated product 2c
also being isolated. Likewise, the reaction also proceeded in good
yields with deactivated heterocyclic amine such as 4-aminopyri-
dine (Table 2, entry 9). The reaction was very slow with 4-nitroan-
iline, and required 72 h for completion, giving a good yield of 85%
(Table 2, entry 17).
Four 3,7-diarylaminocholestane derivatives 3a, 3b, 3h and 3i
were examined for their antimicrobial activities against nine of
Gram-positive strains, such as Staphylococcus aureus ATCC6538P,
S. aureus ATCC25923, S. aureus giorgio, S. aureus 77, S. aureus 241,
Staphylococcus epidermidis 887E, Micrococcus luteus ATCC9341,
Bacillus subtilis ATCC6633, Bacillus licheniformis EMR and three of
Gram-negative strains, such as Escherichia coli ATCC25922, Salmo-
nella typhimurium ATCC13311, Klebsiella pneumoniae 2011E as
described previously [25,26]. Among the tested 3,7-diarylamino
Acknowledgment
This research was supported by the KNU Research Fund, 2013.
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.steroids.2014.
06.018.
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